Several solute carrier proteins (SLC) are expressed in the intestine to absorb nutrients and xenobiotics. In spite of their physiological importance, carrier proteins have not been systematically employed as targets to increase oral drug bioavailability. The current proposal aims to close this gap by focusing on one of the few pharmacologically important transporters in the gastrointestinal tract: the human apical sodium-dependent bile acid transporter (hASBT). hASBT reabsorbs over 12 grams of bile acids daily, suggesting hASBT's tremendous capacity to serve as a drug or prodrug target for absorption. Preliminary data shows that drugs may be conjugated with a bile acid's C-24 carboxylate to yield prodrugs that are translocated by hASBT and increase drug bioavailability. Acyclovir's bioavailability was doubled in rats using a prodrug that targets hASBT. We hypothesize that a systematic structure-activity approach can be used to elucidate the chemistry space of the bile acid's C-24 side chain region that permits bile acid conjugates to be transported by hASBT. The aim of this proposal is to develop a comprehensive and predictive three-dimensional quantitative structure-activity relationship (3D-QSAR) model for the hASBT substrate requirements of bile acid conjugates. 3D-QSAR development will be achieved by synthesizing several congeneric series of bile acid conjugates that systematically span a diverse chemistry space in the C-24 side chain region. Conjugates will be assayed for hASBT-mediated transport and inhibition using in vitro cell culture. A novel molecular modeling approach that considers conformation distribution patterns will be used in 3D-QSAR development. In vivo evaluation of selected bile acid conjugates will be performed in rats, to scale the in vitro 3D-QSAR model to an in vivo 3D-QSAR model. This proposal represents a chemistry-based approach to assess hASBT functioning and complements ongoing biophysical studies. The long-term goal of this research is to use the developed 3D-QSAR model to rationally select parent drugs and their resulting prodrugs for successful hASBT targeting. This systematic and progressive approach will serve as a prototypical method to elucidate the substrate requirements of other solute carrier (SLC) proteins.